Bottom Line:
The effect could be mimicked by purified PAMPs, the most potent of which were curdlan and zymosan, β-(1,3)-glucan-containing polymers that are recognized by Dectin-1.Type B T cells have been shown to escape thymic tolerance and to transfer pathology in an autoimmune disease model.The induction of type B responses by gram-negative bacteria provides a mechanism by which autoreactive T cells may be produced during infection.

fig04: Salmonella and curdlan-induced peptide presentation to type B (11A10) T cells is blocked by anti-Dectin-1 Ab and laminarin. (A) BMDCs were either directly exposed to HKST or preincubated for 1 h with anti-Dectin-1 (2A11) Ab (25 μg/mL), laminarin (500 μg/mL), or anti-Dectin-1 Ab 2A11 (25 μg/mL) and laminarin (500 μg/mL) combined. After 24 h, presentation of HEL46–61 peptide to type B hybridoma (11A10) cells was measured by IL-2 production as described in Figure1. Data are shown as mean ± SEM of 12 samples pooled from four independent experiments. (B) BMDCs were treated as in (A), but exposed to curdlan (100 μg/mL) in place of HKST. Data are shown as mean ± SEM of nine samples pooled from four independent experiments. (C) Comparison of the efficiency of combined laminarin and anti-Dectin Ab blocking upon curdlan and HKST induced peptide presentation to type B T cells plotted as paired datasets. p-Values were calculated using Student's paired t-test.

Mentions:
To confirm the interaction, we also employed blocking with a Dectin-1-specific Ab reagent (2A11) and laminarin, a soluble β-(1,3)-glucan. As shown in Figure4B, the increase in presentation to type B T cells induced by curdlan was significantly blocked by the anti-Dectin-1 Ab 2A11 (63% inhibition), laminarin (55% inhibition), and anti-Dectin-1 Ab and laminarin in combination (66% inhibition). These reagents also inhibited the response induced by S. Typhimurium where anti-Dectin-1 Ab, laminarin, and combined treatment reduced the presentation to type B T cells by 55, 42, and 61%, respectively (Fig.4A). In all experiments, after stimulation with either curdlan or S. Typhimurium, blocking with anti-Dectin-1 Ab and laminarin alone or in combination resulted in reduced presentation to type B T cells. As the blocking with anti-Dectin-1 Ab alone is similar for stimulation with S. Typhimurium and curdlan (55 and 63%), this suggests that a major component of the stimulation by S. Typhimurium is via Dectin-1. To visualize the results of individual experiments, the combined blocking with 2A11 and laminarin are depicted graphically (Fig.4C). In all cases, blocking resulted in reduced presentation to type B T cells. Together, these studies show that Dectin-1 is expressed on BMDCs and that it can recognize β-(1,3)-glucan present on S. Typhimurium, curdlan, and zymosan. This recognition results in enhanced presentation of peptide Ag to type B T cells.

fig04: Salmonella and curdlan-induced peptide presentation to type B (11A10) T cells is blocked by anti-Dectin-1 Ab and laminarin. (A) BMDCs were either directly exposed to HKST or preincubated for 1 h with anti-Dectin-1 (2A11) Ab (25 μg/mL), laminarin (500 μg/mL), or anti-Dectin-1 Ab 2A11 (25 μg/mL) and laminarin (500 μg/mL) combined. After 24 h, presentation of HEL46–61 peptide to type B hybridoma (11A10) cells was measured by IL-2 production as described in Figure1. Data are shown as mean ± SEM of 12 samples pooled from four independent experiments. (B) BMDCs were treated as in (A), but exposed to curdlan (100 μg/mL) in place of HKST. Data are shown as mean ± SEM of nine samples pooled from four independent experiments. (C) Comparison of the efficiency of combined laminarin and anti-Dectin Ab blocking upon curdlan and HKST induced peptide presentation to type B T cells plotted as paired datasets. p-Values were calculated using Student's paired t-test.

Mentions:
To confirm the interaction, we also employed blocking with a Dectin-1-specific Ab reagent (2A11) and laminarin, a soluble β-(1,3)-glucan. As shown in Figure4B, the increase in presentation to type B T cells induced by curdlan was significantly blocked by the anti-Dectin-1 Ab 2A11 (63% inhibition), laminarin (55% inhibition), and anti-Dectin-1 Ab and laminarin in combination (66% inhibition). These reagents also inhibited the response induced by S. Typhimurium where anti-Dectin-1 Ab, laminarin, and combined treatment reduced the presentation to type B T cells by 55, 42, and 61%, respectively (Fig.4A). In all experiments, after stimulation with either curdlan or S. Typhimurium, blocking with anti-Dectin-1 Ab and laminarin alone or in combination resulted in reduced presentation to type B T cells. As the blocking with anti-Dectin-1 Ab alone is similar for stimulation with S. Typhimurium and curdlan (55 and 63%), this suggests that a major component of the stimulation by S. Typhimurium is via Dectin-1. To visualize the results of individual experiments, the combined blocking with 2A11 and laminarin are depicted graphically (Fig.4C). In all cases, blocking resulted in reduced presentation to type B T cells. Together, these studies show that Dectin-1 is expressed on BMDCs and that it can recognize β-(1,3)-glucan present on S. Typhimurium, curdlan, and zymosan. This recognition results in enhanced presentation of peptide Ag to type B T cells.

Bottom Line:
The effect could be mimicked by purified PAMPs, the most potent of which were curdlan and zymosan, β-(1,3)-glucan-containing polymers that are recognized by Dectin-1.Type B T cells have been shown to escape thymic tolerance and to transfer pathology in an autoimmune disease model.The induction of type B responses by gram-negative bacteria provides a mechanism by which autoreactive T cells may be produced during infection.